Oligosaccahrides are important components of a variety of different types of biological molecules, and are involved in antigenic recognition and cell—cell interactions. In many cases, bio-molecules require conjugation with a carbohydrate component in order to be fully functional. In order to enable investigation of the biological function, and to exploit the exquisite biochemical and antigenic specificity of oligosaccharides, it is essential to have access to highly defined, specific synthetic oligosaccharides. Therefore achieving efficient, cost-effective synthesis of oligosaccharides and glycoconjugates by either solution or solid phase methods is of the utmost importance.
This task is enormously complicated by the complexity of oligosaccharides. Because of the number of sites which can carry substituents, and the number of possible ways in which two saccharide molecules can be linked, the number of permutations is enormously high.
In naturally-occurring oligosaccharides D-glucose, D-galactose L-fucose, D-mannose, D-glucosamine and D-galactosamine are among the most common sugar residues. To construct oligosaccharides and carbohydrates conjugates using these sugars, current methodologies require long, protracted syntheses, involving synthesis of as many as one hundred different specially-protected sugar donors in order to cover adequately all the possible permutations of glycosidic link formation (eg. 1-3, 1-4), link type (eg. α or β) and to include all possible branching points in the oligosaccharide.
Orthogonal protection of bi-functional molecules has been a widely used technique in organic chemistry, which provided general building blocks for selected syntheses. However, orthogonal protection in the case of molecules with a greater degree of functionalisation is quite rare. Our technology involves penta-functional monosaccharide building blocks, which require a much higher level of chemical specificity to attain the appropriate orthogonality.
Orthogonal protection has been defined by Merrifield as follows:                “The principle of orthogonal stability requires that only those protecting functions should be used that can be cleaved under different reaction conditions without affecting the other functions present” (Merrifield, 1977)        
Orthogonal protecting strategies and conditions are reviewed in the textbook, “Protecting Groups in Organic Synthesis”, by Green and Wicks (3rd edition). Although the use of orthogonal protection would greatly facilitate carbohydrate related synthesis, there has been limited success in devising suitable protecting groups and methods.
Wong et al. synthesised a universal building block with chloroacetyl, p-methoxybenzyl, levulinyl and tert-butyldiphenylsilyl protecting groups, selectively removably with sodium bicarbonate, trifluoroacetic acid, hydrazine and hydrogen fluoride-pyridine respectively, on a galactopyranose ring with an aryl-thio leaving group at the glycosidic position. This building block was used solely to syntheses a 6-hexanate glycoside. The subsequent recombinant oligosaccharide library formation focused on using the 6-hexane derivatised building block which exhibits only four degrees of orthogonality (Wong et al, 1998).
Similarly Kunz and coworkers synthesized an orthogonally protected D-glucopyranose derivative, but synthetic manipulations were only performed on the aglycon. These authors describe orthogonal protection of hydroxyl groups on a monosaccharide linked at C1 via a thioglycoside group to a solid support or to a succinimide moiety. In this case the protecting groups are acetyl or methy at C2, ally at C3, ethoxyethyl at C4, the tert-butyldiphenylsilyl at C6. The thioglycoside anchor functionalized in the side-chain is stated to be crucial. Again there is no suggestion that this protection system can be used for substituted sugars. Kunz's orthogonally-protected building block was not used for clycosylation or construction of glycoconjugates or neo-glycoconjugates, by directly attaching functionalities to the pyranose ring (Wunberg et al. 1998).
In our earlier International Patent Applications No. PCT/AU97/00544, No. PCT/AU98/00131 and No. PCT/AU98/00808, we described protecting and linking groups which enabled oligosaccharides and aminooligosaccharides to be synthesised using solid phase methods of the type which for many years have been used in peptide synthesis. In addition the protecting groups, described therein were useful for solution-phase synthesis. The entire disclosures of these specifications are incorporated herein by this reference.
We have now devised new types of building blocks which greatly facilitate the synthesis of oligosaccharides and glycoconjugates, using orthogonally-protected saccharide building blocks with five degrees of orthogonally. These building blocks contain a leaving group or latent leaving group at the glycosidic position, and another four orthogonally-protected functional groups around the carbohydrate ring.
Using our approach with six universal building blocks based on six of the most common naturally occurring sugars, any one of the one hundred sugars referred to above may be quickly synthesized in a facile manner, using simple, well-known protecting group chemistry. The years of work and complex protection strategies required to produce these one hundred building blocks by previously-available methods can be avoided by use of our six universal building blocks, which do not require a high level of skill to use, and enable one to achieve the synthesis of a specific desired oligosaccharide or glycoconjugate much faster and most efficiently than previously possible.